The use of nucleic acid hybridization probes for bioassays is well known. One of the early papers in the field directed to assays for DNA is Gillespie, D. and Spiegelman, S., A Quantitative Assay for DNA-RNA Hybrids with DNA Immobilized on a Membrane, J. Mol. Biol. 12:829-842 (1965). In general terms such an assay involves separating the nucleic acid polymer chains in a sample, as by melting, fixing the separated DNA strands to a nitrocellulose membrane, and then introducing a probe sequence which is complementary to a unique sequence of the material being sought, the "target" material, and incubating to hybridize probe segments to complementary target segments, if targets are present. Non-hybridized probes are removed by known washing techniques, and then the amount of probe remaining is determined by one of a variety of techniques outlined below which provides a measurement of the amount of targets in the sample.
A more recently developed form of bioassay that uses nucleic acid hybridization probes involves a second probe, often called a "capture probe." Ranki, M., Palva, A., Virtanen M., Laaksonen, M., and Soderlund, H., Sandwich Hybridization as a convenient Method for the Detection of Nucleic Acids in Crude Samples, Gene 21:77-85 (1983); Syvanen, A.-C., Laaksonen, M., and Soderlund, H., Fast Quantification of Nucleic Acid Hybrids by Affinity-based Hybrid Collection, Nucleic Acids Res. 14:5037-5048 (1986). A capture probe contains a nucleic acid sequence which is complementary to the target, preferably in a region near the sequence to which the radioactively labeled probe is complementary. The capture probe is provided with a means to bind it to a solid surface. Thus, hybridization can be carried out in solution, where it occurs rapidly, and the hybrids can then be bound to a solid surface. One example of such a means is biotin. Langer, P. R., Waldrop, A. A. and Ward, D. C., Enzymatic Synthesis of Biotin-Labeled Polynucleotides: Novel Nucleic Acid Affinity Probes, Proc. Natl. Acad. Sci. USA 78:6633-6637 (1981). Through biotin the capture probe can be bound to streptavidin covalently linked to solid beads.
The present invention is directed to the methods and means, including assays and pharmaceutical kits containing requisite reagents and means, for detecting in an in vitro or ex vitro setting the presence of nucleic acid species.
It is a goal in this art to detect various nucleic acid sequences in a biological sample, in which the said sequences, as so-called target sequences, are present in small amounts relative to its existence amongst a wide variety of other nucleic acid species including RNA, DNA or both. Thus, it is desirable to detect the nucleic acid encoding polypeptides that may be associated with pathological diseases or conditions, such as, for example, RNA of the human immunodeficiency virus. In addition to the detection of nucleic acids encoding the proteins of such viral particles, it is desirable to detect other nucleic acids characteristic of a pathological disease or condition such as a defective gene, as in the case of hemophilia. It is also desirable to detect other nucleic acids whose presence in the sample indicates that the organism is able to resist the action of a drug, such as an antibiotic.
Several approaches have been used for detecting the probe. One is to link a readily detectable reporter group to the probe. Examples of such reporter groups are fluorescent organic molecules and .sup.32 P-labeled phosphate groups. These detection techniques have a practical limit of sensitivity of about a million targets per sample.
A second approach is to link a signal generating system to the probe. Examples are enzymes such as peroxidase. Probes are then incubated with a color-forming substrate. Leary, J. J., Brigati, D. J. and Ward, D. C., Rapid and Sensitive Colorimetric Method for Visualizing Biotin-Labeled DNA Probes Hybridized to DNA or RNA Immobilized on Nitrocellulose: Bio-Blots, Proc. Natl. Acad. Sci. USA 80:4045-4049 (1983). Such amplification reduces the minimum number of target molecules which can be detected. As a practical matter, however, nonspecific binding of probes has limited the improvement in sensitivity as compared to radioactive tagging to roughly an order of magnitude, i.e., to a minimum of roughly 100,000 target molecules.
Yet another approach is to make many copies of the target itself by in vivo methods. Hartley, J. L., Berninger, M., Jessee, J. A., Bloom, F. R. and Temple, G. S., Bioassay for Specific DNA Sequences Using a Non-Radioactive Probe, Gene 49:295-302 (1986). This can also be done in vitro using a technique called "polymerase chain reaction" (PCR). This technique was reported in Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G. T., Erlich, H. A., and Arnheim, N., Enzymatic Amplification of Beta-globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia, Science 230:1350-1354 (1985); Saiki, R. K., Gelfand, D. H. Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A., Primer-directed Enzymatic Amplification of DNA With a Thermostable DNA Polymerase, Science 239:487-491 (1988); Erlich, H. A., Gelfand, D. H., and Saiki, R. K., Specific DNA Amplification, Nature 331:461-462 (1988), and Mullis et al., European Patent Application Publication Nos. 200362 and 201184 (see also U.S. Pat. Nos. 4,683,195 and 4,683,202). In PCR, the probe is complementary only to the beginning of a target sequence but, through an enzymatic process, serves as a primer for replication of an entire target. Each repetition of the process results in another doubling of the number of target sequences until a large number, say, a million copies, of the target are generated. At that point detectable probes, e.g., radioactively labeled probes, can be used to detect the amplified number of targets. The sensitivity of this method of target amplification is generally limited by the number of "false positive signals" generated, that is, generated segments that are not true copies of the target. Nonetheless, this method is quite sensitive. The procedure requires at least two nucleic acid probes and has three steps for a single cycle. This procedure is cumbersome and not always reliable.
Yet another method for amplification is to link to the probe an RNA that is known to be copied in an exponential fashion by an RNA-directed RNA polymerase. An example of such a polymerase is bacteriophage Q-beta replicase. Haruna, I., and Spiegelman, S., Autocatalytic Synthesis of a Viral RNA In Vitro, Science 150:884-886 (1965). Another example is brome mosaic virus replicase. March et al., POSITIVE STRAND RNA VIRUSES Alan R. Liss, New York (1987). In this technique, the RNA serves as a template for the exponential synthesis of RNA copies by a homologous RNA-directed RNA polymerase. The amount of RNA synthesized is much greater than the amount present initially. This amplification technique is disclosed in Chu, B. C. F., Kramer, F. R., and Orgel, L. E., Synthesis of an Amplifiable Reporter RNA for Bioassays, Nucleic Acids Res. 14:5591-5603 (1986); Lizardi, P. M., Guerra, C. E., Lomeli, H., Tussie-Luna, I. and Kramer, F. R., Exponential Amplification of Recombinant-RNA Hybridization Probes, Bio/Technology 6:1197-1203 (October, 1988), which is incorporated herein by reference and is attached hereto in manuscript form [hereinafter referred to as "Lizardi et al."]; published European Patent Application 266,399 (EP Application No. 87903131.8). After non-hybridized probes are removed by washing, the RNA polymerase is used to make copies of the replicatable RNA. According to the disclosure of published European Patent Application No. 266,399, replication of the RNA may take place while the RNA is linked to the probe. Alternatively, the replicatable RNA may be separated from the remainder of the probe prior to replication. That application also discloses a variety of chemical links by which a probe sequence can be joined to a replicatable RNA. In addition, it discloses that the probe sequence may be part of a replicatable RNA, as described in Miele, E. A., Mills, D. R., and Kramer, F. R., Autocatalytic Replication of a Recombinant RNA, J. Mol. Biol. 171:281-295 (1983). That European application also discloses that such recombinant RNAs must be able to hybridize specifically with the target sequence as well as to retain their ability to serve as a template for exponential replication by an appropriate RNA-directed RNA polymerase, as is demonstrated in the results obtained by Lizardi et al., supra.
Replication of RNA, as opposed to target amplification using PCR, can be done in a single step. In that step one can synthesize as many as a billion copies of the replicatable RNA that was joined to the probe in as little as twenty minutes, which theoretically could lead to detection of a single target molecule. However, in practice the sensitivity of this type of probe replication is limited by the persistence of nonspecifically bound probes. Nonspecifically bound probes will lead to replication just as will probes hybridized to targets.
A major problem in the implementation of bioassays that employ hybridization technology coupled to signal amplification systems is the background signal produced by nonspecifically bound probe molecules. These background signals introduce an artificial limit on the sensitivity of bioassays. In conventional bioassays this problem is sometimes alleviated by the utilization of elaborate washing schemes that are designed to remove nonspecifically bound probes. These washing schemes inevitably add to the complexity and cost of the assay.
As a means to reduce the background noise level of assays employing probes linked to replicatable RNA by covalently joined linking moieties, European Patent Application No. 266,399 discloses what it refers to as "smart probes," that is, probes whose linked RNA is said not to serve as a template for replication unless and until the probe has hybridized with a target sequence. In that application two embodiments are disclosed for smart probes.
In a first embodiment in that application, the smart probe comprises a probe portion consisting of about 75-150 deoxynucleotides, made by in vitro or in vivo methods known in the art. The smart probe also comprises a recombinant, replicatable RNA containing an inserted heterologous sequence of about 10-30 nucleotides, made by, e.g., the method of Miele, E. A., Mills, D. R., and Kramer, F. R., Autocatalytic Replication of a Recombinant RNA. J. Mol. Biol. 171:281-295 (1983). Joining those two portions at their 5' ends is a linking moiety of the formula --O(PO.sub.2)NH(CH.sub.2).sub.a SS(CH.sub.2).sub.b NH(PO.sub.2)O--, where a and b are each 2 to 20. Furthermore, the sequence at the 3' end of the DNA portion of the smart probe is capable of being (and very likely to be) hybridized to the heterologous sequence of the RNA portion of the smart probe. The enzyme ribonuclease H is said to be capable of cleaving the RNA portion of smart probes which have not hybridized to targets, but not be capable of cleaving the RNA portion of smart probes which have hybridized to targets, because when the probe sequence in the DNA portion of a smart probe is bound to its target, it is said to be incapable of also being hybridized to the heterologous sequence in the RNA portion of the smart probe, thereby providing a way to eliminate nonspecifically bound probes prior to amplification. Amplification via RNA replication is said to optionally include the preliminary step of cleaving the disulfide bond in the linking moiety.
In that embodiment, cleavage of probes not hybridized to targets is said to be possible for ribonuclease H, because the 3' end of the DNA portion of the smart probe (which contains the probe sequence) is hybridized to the recombinant replicatable RNA portion, presumably thereby providing a site wherein ribonuclease H can cleave the RNA and render it inoperative as a template for amplification by an RNA-directed RNA polymerase.
In the other embodiment of a smart probe disclosed in published European Patent Application 266,399, there is a probe portion, a linking moiety, and a replicatable RNA portion, linked as described above. Here, however, the probe portion comprises not only a probe segment of 50-150 nucleotides, but also additional segments, called "clamp" segments, on either side of it, that is, a 5'-clamp segment and a 3'-clamp segment, each of about 30-60 nucleotides. Each clamp segment is said to hybridize with a segment of the replicatable RNA portion, rendering the RNA inactive as a template for replication, unless and until the probe is hybridized with a target. That hybridization causes the clamps to release, thereby rendering the RNA replicatable, either directly or after optional cleavage of the disulfide bond.
The smart probes disclosed in published European Patent Application No. 266,399 comprise a somewhat complicated linking moiety containing a weakly covalent and rather easily dissociable disulfide linkage. Disulfide bonds readily dissociate under reducing conditions. The two versions of smart probes disclosed in that application rely on distant intramolecular interactions to render the probe smart. This is a disadvantage which makes such probes difficult to design, particularly since distant interactions are not well understood. The second version, reported above, has a further complication that it utilizes two distant clamps which must displace a set of relatively strong neighboring compliments. And, the design depends on both distant clamps hybridizing or none, which makes design very difficult.
An object of the present invention is a simple molecular allosteric switch that renders a nucleic acid hybridization probe smart, that is, capable, in an appropriate assay, of generating a signal only if the probe is hybridized to a target sequence.
It is a further object of this invention to couple the activity of a signal generating system to the state of such a switch.
It is yet another object of this invention to develop probes containing such an allosteric switch that are linked to any of a number of different signal generating systems whose activity is dependent on the state of the switch.
It is another object of this invention to develop assays of improved sensitivity that utilize the above constructs, as well as kits for performing such assays.